GFP Expressing C57BL/6 Mouse Primary Pulmonary Artery Smooth Muscle Cells

Description

Selecting a reporter-expressing primary cell model requires confirming the species, strain, vascular tissue source, cell identity, reporter type, passage, and culture requirements. These factors help researchers determine whether the supplied cells match their planned primary cell culture and reporter-based laboratory workflow.

Luciferase-Expressing C57BL/6 Mouse Primary Pulmonary Artery Smooth Muscle Cells are isolated from the pulmonary artery of pathogen-free laboratory C57BL/6 mice. The cells are grown in gelatin pre-coated tissue culture flasks with Complete Growth Medium.

The Firefly luciferase-expressing cells are developed through transduction with Firefly luciferase lentiviral particles. Luciferase is used in bioluminescence and is distinct from GFP and RFP fluorescent proteins.

Cells are harvested at passages 2–3 and cryopreserved in vials. Each frozen vial contains 0.5 × 10⁶ cells. The mouse primary pulmonary artery smooth muscle cells are characterized by immunofluorescent staining with an α-smooth muscle actin antibody (A2547, Sigma).

The cells are reported negative for bacteria, yeast, fungi, and mycoplasma. Under the specified primary cell culture conditions, they can be expanded for 2–3 passages at a recommended split ratio of 1:2. Repeated freezing and thawing of the cells are not recommended.

Specifications

Features

• Primary pulmonary artery smooth muscle cells isolated from pathogen-free laboratory C57BL/6 mice
• Firefly luciferase expression introduced through lentiviral transduction
• Harvested and cryopreserved at passages 2–3
• Supplied as a frozen vial containing 0.5 × 10⁶ cells
• Characterized by α-smooth muscle actin immunofluorescent staining using antibody A2547, Sigma
• Reported negative for bacteria, yeast, fungi, and mycoplasma
• Expandable for 2–3 passages at a recommended split ratio of 1:2 under specified conditions
• Cultured on vessels coated with CB6950 Gelatin-Based Coating Solution
• Shipped frozen on dry ice and transferred to liquid nitrogen for long-term storage

Applications

These luciferase-expressing C57BL/6 mouse primary pulmonary artery smooth muscle cells are intended for laboratory workflows requiring the documented combination of a mouse pulmonary artery source, primary smooth muscle cell identity, and Firefly luciferase expression.

• Primary pulmonary artery smooth muscle cell culture
• Laboratory research using C57BL/6 mouse vascular smooth muscle cells
• Firefly luciferase reporter-based research
• Bioluminescence-related laboratory research using luciferase-expressing cells
• α-Smooth muscle actin immunofluorescent staining
• Preparation of primary cells for cell-based assays under the supplied culture protocol
• Preparation of cultured primary cells for staining or FACS analysis under the supplied protocol

Specific experimental design, controls, reporter detection conditions, and assay validation should be established by the investigator. The supplied files do not provide product-specific Luciferase signal intensity or expression-stability data.

Cell Culture Workflow

Culture Preparation

• Perform all cell culture procedures in a biosafety cabinet.
• Use aseptic technique to prevent microbial contamination.
• Sterile filter culture media, supplements, and reagents through a 0.2 μm filter.
• Prewarm only the amount of culture medium required for immediate use to 37°C.
• Use 5 mL of medium for a T25 flask and 8–10 mL for a T75 flask when recovering frozen cells, changing medium, or splitting cells.
• Repeated prewarming of complete culture medium is not recommended.

Gelatin Coating

• Coat sterile culture dishes or flasks with CB6950 Gelatin-Based Coating Solution for 2 minutes.
• Aspirate the excess coating solution before seeding cells.

Recovery from a Cryovial

1. Thaw the cryovial rapidly in a 37°C water bath for less than 1 minute or until only a few ice pieces remain.
2. Immediately remove the vial and wipe the exterior with 70% ethanol.
3. Transfer the cells into a sterile 15 mL conical centrifuge tube containing 5 mL of prewarmed culture medium.
4. Rinse the vial with an additional 0.5–1 mL of medium to collect remaining cells.
5. Centrifuge at 200 × g for 5 minutes to remove residual DMSO.
6. Carefully aspirate the supernatant and resuspend the cell pellet in 5 mL of Complete Growth Medium.
7. Seed the resuspended cells into a T25 flask pre-coated with CB6950 Gelatin-Based Coating Solution.
8. Place the flask in a humidified 37°C, 5% CO₂ incubator and leave the culture undisturbed for 12–16 hours.
9. Change the medium on the second day and every 24 hours thereafter.
10. Use 5 mL of medium for a T25 flask and 10 mL for a T75 flask during daily medium changes.
11. Check the cells daily under a microscope for confluence and appropriate morphology.

Primary Cell Expansion

1. Remove the culture medium and wash the cells twice at room temperature with 1X PBS without calcium and magnesium.
2. Incubate cells with CB6914 0.25% Trypsin-EDTA Solution prewarmed to 37°C for 3–5 minutes.
3. Use 0.5–1 mL of Trypsin-EDTA for a T25 flask or 2 mL for a T75 flask.
4. Once the cells have detached, immediately add 5–10 mL of culture medium containing fetal bovine serum to neutralize the trypsin.
5. Gently pipette the suspension up and down several times.
6. Seed the cells into fresh gelatin-coated flasks or plates at the recommended 1:2 split ratio.
7. Return the culture vessels to a humidified incubator at 37°C with 5% CO₂.
8. Change the medium on the second day and every 24 hours thereafter.
9. Before each medium change, pre-wash the cells twice with 1X PBS without calcium and magnesium.
10. Check cell confluence and morphology daily under a microscope.

Preparation for Experiments

• After cells reach confluence, cultures may be allowed to overgrow for 24 hours before cell-based assays, staining, FACS analysis, or other designed experiments.
• During this period, change the medium daily and pre-wash the cells twice with 1X PBS between medium changes.
• Before an experiment, cells may be filtered through a 40 μm cell strainer (BD 352340) to remove cell clumps and most dead or floating large cells.

Cell Freezing Procedure

• Wash adherent cells 2–3 times with sterile 1X PBS without calcium and magnesium.
• Incubate cells with prewarmed CB6914 0.25% Trypsin-EDTA for 3–5 minutes.
• Use 0.5–1 mL of Trypsin-EDTA for a T25 flask or 2–3 mL for a T75 flask during the freezing procedure.
• Neutralize the trypsin with 10 mL of culture medium containing fetal bovine serum.
• Transfer the cell suspension to a centrifuge tube and centrifuge at 200 × g for 5 minutes.
• Remove the supernatant and add 1 mL of CB6916 Freezing Medium per vial.
• Place the vials in a Nalgene “Mr. Frosty” freezing container filled with 100% isopropanol at −80°C for 6–12 hours.
• Transfer the vials to a liquid nitrogen tank for long-term storage.

Quality Control

Storage and Handling

• Cryopreserved cells are shipped frozen on dry ice.
• Upon receipt, promptly transfer the frozen vial from the dry ice shipping container to a liquid nitrogen storage tank.
• If the cells will be thawed on the day of receipt, they may remain on dry ice until thawing begins.
• Do not use −20°C or −80°C as the long-term storage condition for the supplied cryopreserved cells.
• The protocol uses −80°C only for the controlled initial freezing step of 6–12 hours before transfer to liquid nitrogen.
• Repeated freezing and thawing of cells are not recommended.
• Handle animal cells with caution because no testing procedure can completely guarantee the absence of infectious agents.
• Follow institutional biosafety procedures when handling and disposing of cell culture products.

Safety and Intended Use

For Research Use Only. These cells are not authorized for human use or for in vitro diagnostic or therapeutic procedures.

The supplied cell culture products are not classified as hazardous under the safety information provided. Appropriate laboratory safety procedures, eye protection, chemical-resistant gloves, and applicable institutional practices should be followed when handling the product.

Documents

Documents

C57-6083GFP

Cell-Culture-Protocol-CB

1708462493

1698246847

Frequently Asked Questions